AMP-activated protein kinase (AMPK) is a cellular energy sensor whose activity responds to AMP concentration ([AMP]). An agent that activates AMPK in cells is 5-aminoimidazole-4-carboxamide-1-riboside (AICAriboside). Phosphorylated AICAriboside or AICAribotide (ZMP) is an AMP analog. It is generally assumed that ZMP accumulation does not alter [AMP]. Additionally, the effect of AICAriboside on AMPK activity of the heart is uncertain. Two hypotheses were tested in the isolated mouse heart: one, sufficient [ZMP] forms to increase AMPK activity; and, two, [ZMP] accumulation increases [AMP]. Perfusion of isolated mouse hearts with Krebs-Henseleit buffer containing 0.15 to 2 mM [AICAriboside] resulted in [ZMP] of 2 to 8 mM. ZMP accumulation reduced [phosphocreatine], which increased cytosolic [AMP]. In hearts with [ZMP] less than ~3 mM, in vivo AMPK allosteric activity effects of ZMP were observed; AMPK phosphorylation and [AMP] were not increased. With [ZMP] between 3 to 5 mM in vitro AMPK activity and phosphorylation increased with unchanged [AMP]. This occurred in hearts perfused with [AICAriboside] of 0.25 mM for 48 min and 0.5 mM for 24 min. The [ZMP] resulting in 50% AMPK activity (covalent phosphorylation of AMPK) was 4.1 ± 0.6 mM. Hearts with [ZMP] greater than 5 mM displayed increased [AMP] and AMPK activity that was not different from hearts with similar [AMP] with no [ZMP]; the A_0.5 of AMP was 5.6 ± 1.6 µM. Thus, in mouse hearts AICAriboside was metabolized to [ZMP] adequately to increase AMPK activity. Higher [ZMP] also increased cytosolic [AMP], which affects AMPK activity.